Proteins that selectively bind to selected targets by way of non-covalent interaction play a crucial role as reagents in biotechnology, medicine, bioanalytics as well as in the biological and life sciences in general. Antibodies, i.e. immunoglobulins, are a prominent example of this class of proteins. Despite the manifold needs for such proteins in conjunction with recognition, binding and/or separation of ligands/targets, what are currently used are almost exclusively immunoglobulins.
Additional proteinaceous binding molecules that have antibody-like functions are the members of the lipocalin family, which have naturally evolved to bind ligands. Lipocalins occur in many organisms, including vertebrates, insects, plants and bacteria. The members of the lipocalin protein family (Pervaiz, S., & Brew, K. (1987) FASEB J. 1, 209-214) are typically small, secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular-recognition properties: their ability to bind various, principally hydrophobic molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants), their binding to specific cell-surface receptors and their formation of macromolecular complexes. Although they have, in the past, been classified primarily as transport proteins, it is now clear that the lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signalling, and the synthesis of prostaglandins. The lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homoeostasis (reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14 and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).
Lipocalins share unusually low levels of overall sequence conservation, often with sequence identities of less than 20%. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded anti-parallel β-sheet closed back on it to form a continuously hydrogen-bonded β-barrel. This β-barrel forms a central cavity. One end of the barrel is sterically blocked by the N-terminal peptide segment that runs across its bottom as well as three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and encompasses a target-binding site, which is formed by four flexible peptide loops. It is this diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes, each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Flower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, or Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).
Various PCT publications (e.g., WO 99/16873, WO 00/75308, WO 03/029463, WO 03/029471 and WO 2005/19256) disclose how muteins of various lipocalins (e.g. human tear lipocalin) can be constructed to exhibit a high affinity and specificity against a target that is different than a natural ligand of a wild type lipocalin. This can be done, for example, by mutating one or more amino acid positions of at least one of the four peptide loops. In addition, PCT publication WO 2009/095447 teaches methods for generation of lipocalin muteins directed against c-Met.
Generally, kinases are enzymes known to regulate the majority of cellular pathways, especially pathways involved in signal transduction or the transmission of signals within a cell. Because protein kinases have profound effect on a cell, kinase activity is highly regulated. Kinases can be turned on or off by phosphorylation and by binding to activator proteins or inhibitor proteins. Deregulated kinase activity is a frequent cause of disease, particularly cancer where kinases regulate many aspect that control cell growth, movement and death. Many of these genetic defects have been identified as key components of signaling pathways responsible for proliferation and differentiation of cancers. Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab, cetuximab, bevacizumab, imatinib and gefitinib inhibitors have illustrated the interest of targeting this protein class for treatment of selected cancers.
c-Met is the prototypic member of a sub-family of RTKs. The c-Met family is structurally different from other RTK families, and is the only known high-affinity receptor for hepatocyte growth factor (HGF), also called scater factor (SF) (D. P. Bottaro et al., Science 1991, 251: 802-804; L. Naldini et al., Eur. Mol. Biol. Org. J. 1991, 10:2867-2878). In this regard, HGF/SF is the ligand for the c-Met receptor, while c-Met is a receptor tyrosine kinase activated by HGF/SF (Seidel, C., Borset, M., Hjorth-Hansen, H., Sundan, Al., Waage, A., (1998) Role of Hepatocyte Growth Factor and Its Receptor c-Met in Multiple Myeloma Med Oncol 15, 145-53; Brset, M., Seidel, C., Hjorth-Hansen, H., Waage, A., Sundan, A. (1999) The Role of Hepatocyte Growth Factor and Its Receptor c-Met in Multiple Myeloma and Other Blood Malignancies Leukemia & Lymphoma 32, 249-256).
Multiple signaling pathways have been associated with the biological responses mediated by c-Met activation (Abounader, R., et al. (2001) Signaling Pathways in the Induction of c-Met Receptor Expression by its Ligand Scatter Factor/Hepatocyte Growth Factor in Human Glioblastoma, J. Neurochem, 75, 1497-1508). When HGF/SF activates c-Met, the latter in turn may activate a number of kinase pathways, including, but not limited to, the pathway from Ras to Raf to Mek to the mitogen-activated protein kinase ERK1 to the transcription factor ETS1. Co-expression of unaltered c-Met and HGF/SF, as well as activating mutations, are oncogenic (Abounader, R., et al. (2001) Signaling Pathways in the Induction of c-Met Receptor Expression by its Ligand Scatter Factor/Hepatocyte Growth Factor in Human Glioblastoma, J. Neurochem, 75, 1497-1508).
c-Met and HGF are both required for normal mammalian development and have been shown to be particularly important in cell migration, morphogenic differentiation, and organization of the three-dimensional tubular structures as well as growth and angiogenesis (F. Baldt et al., Nature 1995, 376:768-771; C. Schmidt et al., Nature. 1995, 373:699-702; Tsarfaty et al., Science 1994, 263:98-101). While the controlled regulation of c-Met and HGF have been shown to be important in mammalian development, tissue maintenance and repair (Nagayama T, Nagayama M, Kohara S, Kamiguchi H, Shibuya M, Katoh Y, Itoh J, Shinohara Y., Brain Res. 2004, 5; 999(2):155-66; Tahara Y, Ido A, Yamamoto S, Miyata Y, Uto H, Hon T, Hayashi K, Tsubouchi H., J Pharmacol Exp Ther. 2003, 307(1):146-51), their dysregulation is implicated in the progression of cancers.
It also has been known in the art that c-Met plays a role in normal hematopoiesis, and is expressed in various lymphoid and leukemic cell lines. A wide variety of human tumors express both c-Met and HGF/SF and their expression contribute to the malignant progression of gliomas. In addition, overexpression of either HGF or c-Met is found in several cancers, and have been correlated with disease progression and clinical outcome (Ferracini, R., DiRenzo, M. F., Scotlandi, J., Baldini, N., Olivero, M., Lollini, P., Cremona, O., Campanacci, M., Comoglio, P. M. (1995) The Met/HGF Receptor Is Over-Expressed in Human Osteosarcomas and Is Activated By Either a Paracrine or an Autocrine Circuit Oncogene 10, 739-49; Rusciano, D., Lorenzoni, P., Burger, M. M. (1995) Expression of Constitutively Activated Hepatocyte Growth Factor/Scatter Factor Receptor (c-Met) in B16 Melanoma Cells Selected for Enhanced Liver Colonization Oncogene 11, 1979-87). Furthermore, c-Met has been implemented in the development and progression of colon cancer (Herynk, M. H., Stoeltzing, O., Reinmuth, N., Parikh, N. U., Abounader, R., Laterra, J., Radinsky, R., Ellis, L. M., Gallick, G. E. (2003) Down-Regulation of c-Met Inhibits Growth in the Liver of Human Colorectal Carcinoma Cells Cancer Res 63, 2990-6, prostate cancer, Kim, S. J., Johnson, M., Koterba, K., Herynk, M. H., Uehara, H., Gallick, G. E. (2003) Reduced c-Met Expression By an Adenovirus Expressing a c-Met Ribozyme Inhibits Tumorigenic Growth and Lymph Node Metastases of PC3-LN4 Prostate Tumor Cells in an Orthotopic Nude Mouse Model Clin Cancer Res 9, 5161-70), and cancer in other organs (Longati, P., Comoglio, P. M., Bardelli, A. (2001) Receptor Tyrosine Kinases as Therapeutic Targets: the Model of the MET Oncogene Curr Drug Targets 2, 41-55), as well in blood malignancies such as multiple myeloma (Brset, M., Seidel, C., Hjorth-Hansen, H., Waage, A., Sundan, A. (1999) The Role of Hepatocyte Growth Factor and Its Receptor c-Met in Multiple Myeloma and Other Blood Malignancies Leukemia & Lymphoma 32, 249-256). c-Met activation enhances cellular proliferation, migration, morphogenesis, survival (including protection from apoptosis), and protease synthesis, characteristics that are associated with invasive cell phenotype and poor clinical outcomes and drug resistance in cancer patients.
Inappropriate c-Met activation can arise, for example, by so-called ligand-dependent mechanisms (J. G. Christensen, Burrows J. and Salgia R., Cancer Latters. 2005, 226:1-26). In this sense, binding of HGF to c-Met can lead to receptor dimerization or multimerization, phosphorylation of multiple tyrosine residues in the intracellular region, catalytic activation, and downstream signaling. On the other hand, c-Met may also be activated via so-called ligand-independent mechanisms. This activation can be instigated by, for example, receptor over-expression and/or amplification, or paracrine or autocrine activation and/or mutation. In either case, the aberrant signaling pathway driven by inappropriate activation of c-Met is one of the most frequently dysregulated pathways in human cancers, occurs in virtually all types of solid tumors and plays a crucial role in tumorigenesis and metastasis (Birchmeier et al., Nat. Rev. Mol. Cell Biol. 2003, 4:915-925; L. Trusolino and Comoglio P. M., Nat Rev. Cancer. 2002, 2(4):289-300).
Various therapeutic approaches are aimed at the HGF/c-Met pathway. However, no therapeutic methods that possess the features attendant to the therapeutic methods provided by present disclosure have been previously described.
Moreover, with the overexpression and over-activation of c-Met in various cancers being linked to increased proliferation, progression to metastatic disease, and drug resistance (Peruzzi B, Bottaro D P: Targeting the c-met signaling pathway in cancer. Clin Cancer Res 2006, 12(15):3657-3660), the development of a lipocalin mutein that is suitable to assess, in vivo, changes in Met expression would improve the accuracy of diagnosis of c-Met-mediated disease and monitoring of responses to c-Met-targeted therapies.
The need is therefore felt for improved solutions enabling more reliable detection of cells expressing c-Met (e.g. tumorigenic cells), which is as convenient and economical as possible, and this disclosure provides such improved solutions.